CN106665358A - Rapid shoot induction and tissue culture propagation method for platycodon grandiflorum leaves - Google Patents
Rapid shoot induction and tissue culture propagation method for platycodon grandiflorum leaves Download PDFInfo
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- CN106665358A CN106665358A CN201710012158.4A CN201710012158A CN106665358A CN 106665358 A CN106665358 A CN 106665358A CN 201710012158 A CN201710012158 A CN 201710012158A CN 106665358 A CN106665358 A CN 106665358A
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- Prior art keywords
- platycodon grandiflorum
- bud
- leaf
- tissue culture
- quick
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention relates to a rapid shoot induction and tissue culture propagation method for platycodon grandiflorum leaves, which belongs to the technical field of plant regeneration by the tissue culture technique. Specifically, platycodon grandiflorum seeds are first soaked in gibberellin solution, and then disinfected by disinfector and MS basic medium is inoculated with the platycodon grandiflorum seeds for culture, aseptic test-tube plantlets are obtained after seeds germinate to give off seven to ten leaves, the leaves of the aseptic test-tube plantlets are picked, petioles are removed, the leaves are cut into blocks, and thereby cut leaf blocks are obtained; adopted as explants, the cut leaf blocks are transferred into shoot induction medium for culture, and are directly induced to produce differentiated buds without undergoing callus formation and multiplication stages; the obtained differentiated buds are cut off from the bases, transferred into rooting medium and cultured for 30 to 35 days, so that aseptic plantlets are obtained, and after conditions permit, the aseptic plantlets can be transplanted. The cut leaf blocks are utilized as explants to directly carry out induced bud differentiation without undergoing the callus formation and multiplication stages, consequently, the flow is simplified, the plantlet emergence time is shortened, and the propagation coefficient is higher.
Description
Technical field
Plant regeneration field the invention belongs to pass through tissue culture technique, more particularly to a kind of the quick of Leaf of Platycodon Grandiflorum
Lure bud and tissue culture propagation method.
Background technology
Balloonflower root (Platycodon grandiflorus), there have the effect of eliminating the phlegm, relieving sore-throat, antibechic, apocenosis, chest wide to be pleasant, its
Aqueous extract also has antitumaous effect.In recent years, as its health care and medical functions are known from, its price also constantly on
Rise, supply falls short of demand to cause in the market balloonflower root, therefore, the sapling multiplication speed of balloonflower root is improved to meeting need of the market to balloonflower root seedling
Ask significant, will also bring huge economic benefit.
At present, the modes of reproduction of balloonflower root mainly has：Seminal propagation, culturing and transplanting seedlings, cuttage and tissue cultured in vitro, wherein
It is again main modes of reproduction with seminal propagation.Seminal propagation needs reserve seed for planting, choose seeds, carrying out Seed germination, sowing, field management
Etc. step, and except seed needs presoaking and germinating in itself during sowing, the humiture to field also has higher requirements, its emerge when
Between also all at 10-15 days or so, growth cycle is 2-3, and the life-span of seed typically only has 1 year, germination percentage then
Only 70% or so, seminal propagation is also easily caused quality deterioration, therefore method using seminal propagation limits balloonflower root production
Development.And the method for culturing and transplanting seedlings, although the cycle is one year but its step is still comparatively laborious, and to artificial, temperature, water
Point, soil requirement it is higher, be awkward, and yield be less than seminal propagation.Balloonflower root can also carry out cutting propagation, but it belongs to
Perennial root draft, likes the dry environment of low humidity, and using the method easy infection pest and disease damage of cuttage, and rooting rate is low, causes to survive
Rate is not high, therefore with less in production.
Plant tissue cultured in vitro is big using being turned out in a small amount of material short time because not influenceed by external environmental condition
Aseptic nursery stock is measured, therefore is widely used the quick breeding in nursery stock.In traditional group culturation rapid propagating technology, its operating process one
As be：The explant such as blade, stem section → formation callus → callus squamous subculture propagation → callus induction buds sprouting → lure
Lead → rooting culture seedling of taking root.At present, because not high using the inductivity that blade carries out tissue cultures Multiple Buds, the group of balloonflower root
Knit cultured in vitro mainly carried out using the tender stem section of children, stem apex it is fast numerous.But the explant quantity of stem section, stem apex is far below blade
Explant quantity, and, with stem as explant（Such as be free of stipes）Cannot accomplish without the direct induction buds sprouting of callus, culture week
Phase is more long；If the stem section containing stipes can then accomplish without the direct induction buds sprouting of callus, but breeding coefficient is relatively low, only
2 times.
The content of the invention
Regarding to the issue above, bud and tissue culture propagation method are lured the invention provides a kind of the quick of Leaf of Platycodon Grandiflorum, the invention
The induction differentiation of bud is directly carried out using leafcutting as explant, differentiation rate reaches more than 98%, breeding（Propagation）Coefficient reaches
10.15 times, and only needed to 20 days or so from leafcutting to ripe bud is formed, greatly reduce into the bud cycle.
A kind of the quick of Leaf of Platycodon Grandiflorum tissue culture propagation lures bud method, including Seed of Platycodon Grandiflorum is processed and culture acquisition tangerine is sprouted
The step of stalk blade, the step of go handle stripping and slicing to obtain leafcutting gained Leaf of Platycodon Grandiflorum, it is characterised in that：Using leafcutting as
Explant, is transferred in bud inducement cultivation base and is cultivated, and without calli induction and multiplicative stage, directly induction is produced point
Change bud；
Described bud inducement cultivation base is the MS culture mediums of pH=5.8, is added to the working concentration respectively 6- of 1mg/L
Benayl aminopurine（6-BA）, 0.1mg/L methyl α-naphthyl acetate（NAA）, 6.5g/L agar powder and the sucrose of 30g/L；
The condition cultivated in bud inducement cultivation base that is transferred to is：23 ± 2 DEG C of temperature, light application time 12h/d, illumination
The lx of intensity 2000.
Further, the explant cultivates 20d on bud inducement cultivation base and can produce Bud Differentiation.
Further, the size of the leafcutting is 0.5cm × 0.5cm.
The quick of above-mentioned Leaf of Platycodon Grandiflorum tissue culture propagation lures bud method, also including processing Seed of Platycodon Grandiflorum and sprouts culture and obtains
The step of Leaf of Platycodon Grandiflorum, its operating method is：Seed of Platycodon Grandiflorum is soaked in Gibberellins solution, then sterilized dose of sterilization, inoculation
Cultivated in MS basal mediums, when seed is sprouted and grows 7-10 piece leaves, obtained in vitro cuttings, taken its blade.
Further, the concentration of described Gibberellins solution is 250 mg/L, and soak time is 8h.
Further, described disinfectant sterilisation step is：First with the s of 75% alcohol disinfecting 10,1.5 g/L are then used
HgCl2 sterilization 10-15 min.
Using the method for luring bud method to carry out Leaf of Platycodon Grandiflorum tissue culture propagation, it is characterised in that：After quickly being lured bud,
The Bud Differentiation of gained is cut from base portion, is transferred in root media and is cultivated 30-35 days, the aseptic balloonflower root seedling of acquisition, treat condition into
Can be transplanted after ripe.
Further, described root media is the 1/2 MS culture mediums of pH=5.8, is added to working concentration point
Not Wei 0.5 mg/L indolebutyric acid（IBA）, the NAA of 0.5 mg/L, the agar powder of 6.5 g/L and 30 g/L.
Beneficial effects of the present invention：
1st, explant used is to sprout to taking blade and stripping and slicing afterwards to a certain degree Seed of Platycodon Grandiflorum treatment in the method for the invention,
In the process, by seed first in the high concentration Gibberellins solution of 250 mg/L（GA3）Middle immersion 8h is pre-processed, and is broken
Seed dormancy, increases the content of sequestered gibberellin in seed, stratification, while in making the test tube seedling tissue that germination is produced
GA content is higher than mean level, and promotion lures bud；Sterilize afterwards, prevent mould contamination from influenceing test tube seedling growth vigor, through upper
Integrated treatment is stated, percentage of seedgermination is up to more than 95%, and state of growing is good, concentration is kept away with effective combination of pretreatment time
Rosette-stape growth or stem section excessive growth are exempted from, clip blade when length is to 7-10 piece leaves, now test tube seedling growth tends to healthy and strong, together
When blade cell splitting ability it is stronger, go as explant after handle stripping and slicing, abundance, the gibberellin of remaining can continue to promote hair
Bud, is that explant quickly lures bud to lay the foundation.
2nd, the present invention is explant using the in vitro cuttings leafcutting that seed is obtained, with reference to defined medium and hormone
Proportioning, germination culture medium used is the MS culture mediums of pH=5.8, the also NAA and 30g/L of 6-BA, 0.1mg/L comprising 1mg/L
Sucrose, wherein, 6-BA activity it is higher, can promote explant substance in vivo accumulate and cell division, promote bud formation, while
Also can induce callus to occur, the NAA of addition is entered into explant, dredges site of action in company with nutrition stream, specifically
The hormone-content proportioning of 6-BA and NAA, the subsequent affect of gibberellin pre-process in addition in can suppress the formation of callus, accelerate bud
Generation induction, eliminate Callus formation and propagation two incubation times in stage, from leafcutting to formation
Ripe bud（Can be used in the bud/seedling taken root）Only need to 20 days or so, and generally lured by callus again by leafcutting callus long
Leading into bud needs 35-50 days, substantially reduces growth cycle, and the differentiation rate of its blade induced bud reaches more than 98%, to section
Learn research and bring facility with industrial production, it is cost-effective.
3rd, for the tissue cultures of balloonflower root, from the point of view of result of the test and Research Literature, cannot also be done by explant of root
To without the direct induction buds sprouting of callus；With stem as explant（Such as be free of stipes）Also cannot accomplish directly to be induced into without callus
Bud；If the stem section containing stipes can then accomplish without the direct induction buds sprouting of callus, but breeding coefficient is relatively low, only 2 times,
And the inventive method is utilized, its breeding coefficient is higher by about 10% up to 10.15 times.So, the method for the invention breeding coefficient
High energy accesses a large amount of balloonflower root seedlings, is convenient for large-scale production.
Brief description of the drawings
Fig. 1 is that leafcutting is inoculated into seedling differentiation and growing way situation when being cultivated 20 days on bud inducement cultivation base；
Fig. 2 is the situation of taking root that Bud Differentiation is transferred to root media culture 35d；
Fig. 3 is induction situation of the two kinds of combination of regulators of 0.1mg/L 6-BA and 0.05 mg/L NAA to Multiple Buds.
Specific embodiment
With reference to specific embodiment the present invention will be further explained explanation.
Embodiment 1
The configuration of culture medium：
MS basal mediums：MS culture mediums+6.5 g/L of agar powder+sucrose 30 g/L, pH 5.8；
Bud inducement cultivation base：0.1 mg/L of mg/L+NAA of MS culture mediums+6-BA 1+6.5 g/L of agar powder+sucrose
30 g/L, pH 5.8；
Root media composition is the mg/L NAA of+0.5 mg/L IBA of 1/2 MS culture mediums+0.5+g/L of agar powder 6.5
+ sucrose 30 g/L, pH 5.8.
Full, healthy Seed of Platycodon Grandiflorum is chosen, in the gibberellin of 250 mg/L（GA3）8h is soaked in solution, sterilized water is used
Carried out disinfection after flushing, Seed of Platycodon Grandiflorum be placed in 10s is processed in 75% alcohol first, aseptic water washing once, after 1.5 g/
The HgCl of L210-15 min are soaked in solution, with aseptic water washing 4-5 times, the purpose of sterilization is reached.By sterilized seed
It is placed on aseptic filter paper, blots surface moisture, be inoculated on MS basal mediums, above step is grasped on superclean bench
Make.Then by be inoculated with seed MS basal mediums be placed in tissue cultures between cultivated, the condition between tissue cultures is set as：
23 ± 2 DEG C of temperature, light application time daily 12 h and the lx of intensity of illumination 2000.To seed Germination And Seedling, leaf is 7- on seedling for culture
At 10, in vitro cuttings are obtained.
Aseptically, the blade of in vitro cuttings is taken, handle is removed, about 0.5 cm × 0.5 is cut into scalpel
The stripping and slicing of cm sizes, leafcutting is transferred in bud inducement cultivation base, and 20d, leafcutting edge are cultivated between being placed in tissue cultures
Differentiation produces Multiple Buds, and aseptically Multiple Buds are cut from its base portion, is transferred in root media respectively, culture of rootage
30-35d, obtains balloonflower root seedling.Conventional hardening can be carried out afterwards and is transplanted.
The balloonflower root of test example 1 routine tissue culture method
1st, material and treatment
It is examination material with Seed of Platycodon Grandiflorum, seed is put into triangular flask is soaked a few hours with running water first, then with added with washing on a small quantity
The running water immersion 10-15min of clean essence, is rinsed well with running water；Sterilize 1min in the alcohol for adding 75%, is rushed with distilled water
Wash 1-2 times；Seed is transferred in the triangular flask of sterilizing, 0.1% mercuric chloride liquid soaking sterilization 10min is added, aseptic water washing 5-6 is used
It is secondary；Sterilized seed is placed on aseptic filter paper, surface moisture is blotted, is inoculated on MS basal mediums, after the completion of inoculation
Culture in 25 DEG C of incubators is placed on, band seed is sprouted when growing 3-5 piece leaves, you can used as explant.
2nd, program is cultivated
Cultured aseptic seedling is taken out from culture vessel, blade is cut into the fritter of 0.5-1cm, blade face upward, is inoculated in
On callus inducing medium, cultivate 2-3 weeks, you can implant of regarding sb. as an outsider surface forms many callus.Wherein, callus
Culture medium prescription is：MS culture medium+6-BA 0.8-1.5mg/L+IAA 0.3-0.7mg/L+sucrose 20g/L+ agar 5-
7g/L, pH=5.8.
Explant with callus is transferred in bud inducement cultivation base, is cultivated 3-4 weeks, you can the shape from callus
Into many adventitious buds, also known as Multiple Buds.Wherein, bud inducement cultivation based formulas are：MS culture medium+6-BA 0.3-0.5mg/L,
pH=5.8.Multiple Buds are cut from base portion, is placed on same culture medium and is cultivated, larger unrooted test tube seedling can be grown up to.
Unrooted test tube seedling is transferred on root media and is cultivated, culture induces it to take root and grow up to balloonflower root seedling for 3-4 week, then
Can transplant.Wherein, prescription of rooting medium is:1/2 MS culture medium+IAA 0.3-0.5mg/L+IBA 0.01-0.03mg/
L+sucrose 20-25g/L+agar 5.5g/L, pH=5.8.
Embodiment 1 and test example 1 are carried out into overall merit, can complete quickly to sprout using the method for embodiment 1 first, cut from blade
Block is to forming Multiple Buds（Can be used in the bud/seedling taken root）Only need to 20 days or so, and Multiple Buds yield is more, and test example 1
Method needs about 35-50 days by callus induction buds sprouting again by leafcutting callus long, and only culture callus is accomplished by 21-
28 days, for comparing, the method for embodiment 1 is accelerated sprouted, and substantially reduces growth cycle, cost-effective.In addition, such as Fig. 1, real
Apply the gained Multiple Buds of example 1 just had a small amount of root to break up before culture of rootage, and Multiple Buds only have slight vitrification phenomenon, and
Without Furcation defects, vitrification phenomenon has a strong impact on the yield of balloonflower root seedling to the conventional Multiple Buds of test example 1 up to 50%.
Relative to test example 1, the growth of the gained balloonflower root seedling of embodiment 1 is more healthy and strong, and stem, leaf and root physically well develop, and such as schemes
2, average number of being taken root per young plant is 9, a length of 2.5cm of average root, and root hair is more flourishing.
The induction impact analysis of the different growth regulator confrontation Multiple Buds of test example 2
During configuration bud inducement cultivation base, consider balloonflower root and grow and tissue cultures demand, with reference to pre-stage test result, really
Determine two kinds of combinations of growth regulator of 6-BA and NAA, three levels, such as table 1 below are distinguished for two kinds of contents of composition in combination
It is shown.
Table 1：The level design of two factor three
According to the level of two factor three of 6-BA and NAA in table 1, nine treatment experiments in design table 2 below, in addition to culture medium, other
Treatment and condition of culture are same as Example 1, and finally the value-added coefficient and growing state to this nine groups of experiment gained differentiation seedlings enter
Row analysis, wherein, breeding coefficient refers to that can averagely be bred to obtain how many plants of buds with one piece of explant（Seedling）.
Table 2：Each treatment and its differential growth situation
As can be seen from Table 2, the 6-BA of 1mg/L concentration is best suitable for blade differentiation, and comparatively speaking 0.1mg/L NAA are better than
The combination of 0.5mg/L and 0.05mg/L NAA.It is inoculated on the bud inducement cultivation base of 6-BA containing 1mg/L and 0.1mg/L NAA and trains
The differential growth situation for supporting the balloonflower root tests for sterility stripping and slicing of 20d is combined better than other, and growth conditions are good, differentiate a large amount of clumps
Sprout, wherein vitrification phenomenon is slight and has a small amount of root differentiated, such as Fig. 1.Differentiation situation under other combination of regulators
Not ideal, to process as a example by 9, differentiation situation is as shown in figure 3, only very small amount Multiple Buds, and stem development is not healthy and strong enough,
There is lodging, though there are Furcation defects, value-added coefficient is compared to relatively low.
Claims (8)
1. a kind of the quick of Leaf of Platycodon Grandiflorum tissue culture propagation lures bud method, including Seed of Platycodon Grandiflorum is processed and culture acquisition balloonflower root is sprouted
The step of blade, the step of go handle stripping and slicing to obtain leafcutting gained Leaf of Platycodon Grandiflorum, it is characterised in that：Using leafcutting as outer
Implant, is transferred in bud inducement cultivation base and is cultivated, and without calli induction and multiplicative stage, directly induction produces differentiation
Bud；
Described bud inducement cultivation base is the MS culture mediums of pH=5.8, is added to the working concentration respectively 6- of 1mg/L
The agar powder of NAA, 6.5g/L of BA, 0.1mg/L and the sucrose of 30g/L；
The condition cultivated in bud inducement cultivation base that is transferred to is：23 ± 2 DEG C of temperature, light application time 12h/d, illumination
The lx of intensity 2000.
2. the quick of Leaf of Platycodon Grandiflorum tissue culture propagation as claimed in claim 1 lures bud method, it is characterised in that：The explant is in bud
20d is cultivated on inducing culture.
3. the quick of Leaf of Platycodon Grandiflorum tissue culture propagation as claimed in claim 1 lures bud method, it is characterised in that：The leafcutting
Size is 0.5cm × 0.5cm.
4. the quick of Leaf of Platycodon Grandiflorum tissue culture propagation as claimed in claim 1 lures bud method, it is characterised in that：It is described to Seed of Platycodon Grandiflorum
Process and sprout culture obtain Leaf of Platycodon Grandiflorum operating method be：Seed of Platycodon Grandiflorum is soaked in Gibberellins solution, then it is sterilized
Agent is sterilized, and is inoculated into MS basal mediums and is cultivated, and when seed is sprouted and grows 7-10 piece leaves, obtains sterile test tube
Seedling, takes its blade.
5. the quick of Leaf of Platycodon Grandiflorum tissue culture propagation as claimed in claim 4 lures bud method, it is characterised in that：Described gibberellin is molten
The concentration of liquid is 250 mg/L, and soak time is 8h.
6. the quick of Leaf of Platycodon Grandiflorum tissue culture propagation as claimed in claim 4 lures bud method, it is characterised in that：Described disinfectant disappears
Malicious step is：First with the s of 75% alcohol disinfecting 10, then with 1.5 g/L HgCl2Sterilization 10-15 min.
7. using the method for luring bud method to carry out Leaf of Platycodon Grandiflorum tissue culture propagation described in claim 1, it is characterised in that：Carry out quick
After luring bud, the Bud Differentiation of gained is cut from base portion, be transferred in root media and cultivate 30-35 days, the aseptic balloonflower root seedling of acquisition is treated
Can be transplanted after condition maturity.
8. the method for Leaf of Platycodon Grandiflorum tissue culture propagation as claimed in claim 7, it is characterised in that：Described root media is pH=
5.8 1/2 MS culture mediums, are added to working concentration and are respectively the IBA of 0.5 mg/L, the NAA of 0.5 mg/L, 6.5
The sucrose of the agar powder of g/L and 30 g/L.
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| CN201710012158.4A CN106665358A (en) | 2017-01-09 | 2017-01-09 | Rapid shoot induction and tissue culture propagation method for platycodon grandiflorum leaves |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113068611A (en) * | 2021-04-09 | 2021-07-06 | 中国热带农业科学院热带作物品种资源研究所 | Method for rapidly propagating seedlings by utilizing leaves of tetrapanax papyrifera |
| CN114403008A (en) * | 2022-01-28 | 2022-04-29 | 中国科学院近代物理研究所 | Rapid platycodon grandiflorum radiation mutation breeding method |
-
2017
- 2017-01-09 CN CN201710012158.4A patent/CN106665358A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 吴京姬: ""桔梗的组织培养研究"", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 舒娈等: ""桔梗的组织培养"", 《植物资源与环境学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113068611A (en) * | 2021-04-09 | 2021-07-06 | 中国热带农业科学院热带作物品种资源研究所 | Method for rapidly propagating seedlings by utilizing leaves of tetrapanax papyrifera |
| CN114403008A (en) * | 2022-01-28 | 2022-04-29 | 中国科学院近代物理研究所 | Rapid platycodon grandiflorum radiation mutation breeding method |
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Application publication date: 20170517 |